A culture system to study oligodendrocyte myelination processes using engineered nanofibers

Current methods for studying central nervous system myelination necessitate permissive axonal substrates conducive to myelin wrapping by oligodendrocytes. We have developed a neuron-free culture system in which electron-spun nanofibers of varying sizes substitute for axons as a substrate for oligodendrocyte myelination, thereby allowing manipulation of the biophysical elements of axonal-oligodendroglial interactions. To investigate axonal regulation of myelination, this system effectively uncouples the role of molecular (inductive) cues from that of biophysical properties of the axon. We use this method to uncover the causation and sufficiency of fiber diameter in the initiation of concentric wrapping by rat oligodendrocytes. We also show that oligodendrocyte precursor cells display sensitivity to the biophysical properties of fiber diameter and initiate membrane ensheathment before differentiation. The use of nanofiber scaffolds will enable screening for potential therapeutic agents that promote oligodendrocyte differentiation and myelination and will also provide valuable insight into the processes involved in remyelination.     

Evidence for Cooperative Selection of Axons for Myelination by Adjacent Oligodendrocytes in the Optic Nerve

The cellular mechanisms that regulate the topographic arrangement of myelin internodes along axons remain largely uncharacterized. Recent clonal analysis of oligodendrocyte morphologies in the mouse optic nerve revealed that adjacent oligodendrocytes frequently formed adjacent internodes on one or more axons in common, whereas oligodendrocytes in the optic nerve were never observed to myelinate the same axon more than once. By modelling the process of axonal selection at the single cell level, we demonstrate that internode length and primary process length constrain the capacity of oligodendrocytes to myelinate the same axon more than once. On the other hand, probabilistic analysis reveals that the observed juxtaposition of myelin internodes among common sets of axons by adjacent oligodendrocytes is highly unlikely to occur by chance. Our analysis may reveal a hitherto unknown level of communication between adjacent oligodendrocytes in the selection of axons for myelination. Together, our analyses provide novel insights into the mechanisms that define the spatial organization of myelin internodes within white matter at the single cell level.     

Cellular Mechanism of Myelination in the Central Nervous System

A study of myelination with electron microscopy has been carried out on the spinal cord of young rats and cats. In longitudinal and transverse sections the intimate relationship of the growing axons with the oligodendrocytes was observed. Early naked axons appear to be embedded within the cytoplasm and processes of the oligodendrocytes from which they are limited only by the intimately apposed membranes of both elements (axon-oligocytic membrane). In a transverse section several axons are observed to be in a single oligodendrocyte. The process of myelination consists in the laying down, within the cytoplasm of the oligodendrocyte and around the axon, of concentric membranous myelin layers. The first of these layers is deposited at a certain distance (200 to 600 A or more) from the axon-oligocytic membrane. This and all the other subsequently formed membranes have higher electron density and are apparently formed by the coalescence and fusion of vesicles (of 200 to 800 A) and membranes found in large amounts within the cytoplasm of the oligodendrocytes. At an early stage the myelin layers may be discontinuous and some vesicular material may even be trapped among them or between the myelin proper and the axon-oligocytic membrane. Then, when the 8th to 10th layer is deposited, the complete coalescence and alignment of the lamellae leads to the characteristic orderly multilayered organization of the myelin sheath. Myelination in the central nervous system appears to be a process of membrane synthesis within the cytoplasm of the oligodendrocyte and not a result of the wrapping of the plasma membranes as postulated in Geren's hypothesis for the peripheral nerve fibers. The possible participation of Schwann cell cytoplasm in peripheral myelination is now being investigated.     

Cellular mechanism of myelination in the central nervous system

A study of myelination with electron microscopy has been carried out on the spinal cord of young rats and cats. In longitudinal and transverse sections the intimate relationship of the growing axons with the oligodendrocytes was observed. Early naked axons appear to be embedded within the cytoplasm and processes of the oligodendrocytes from which they are limited only by the intimately apposed membranes of both elements (axon-oligocytic membrane). In a transverse section several axons are observed to be in a single oligodendrocyte. The process of myelination consists in the laying down, within the cytoplasm of the oligodendrocyte and around the axon, of concentric membranous myelin layers. The first of these layers is deposited at a certain distance (200 to 600 A or more) from the axon-oligocytic membrane. This and all the other subsequently formed membranes have higher electron density and are apparently formed by the coalescence and fusion of vesicles (of 200 to 800 A) and membranes found in large amounts within the cytoplasm of the oligodendrocytes. At an early stage the myelin layers may be discontinuous and some vesicular material may even be trapped among them or between the myelin proper and the axon-oligocytic membrane. Then, when the 8th to 10th layer is deposited, the complete coalescence and alignment of the lamellae leads to the characteristic orderly multilayered organization of the myelin sheath. Myelination in the central nervous system appears to be a process of membrane synthesis within the cytoplasm of the oligodendrocyte and not a result of the wrapping of the plasma membranes as postulated in Geren's hypothesis for the peripheral nerve fibers. The possible participation of Schwann cell cytoplasm in peripheral myelination is now being investigated.     

Rat optic nerve oligodendrocytes develop in the absence of viable retinal ganglion cell axons.

Retinal ganglion cell axons and axonal electrical activity have been considered essential for migration, proliferation, and survival of oligodendrocyte lineage cells in the optic nerve. To define axonal requirements during oligodendrogenesis, the developmental appearance of oligodendrocyte progenitors and oligodendrocytes were compared between normal and transected optic nerves. In the absence of viable axons, oligodendrocyte precursors migrated along the length of the nerve and subsequently multiplied and differentiated into myelin basic protein-positive oligodendrocytes at similar densities and with similar temporal and spatial patterns as in control nerves. Since transected optic nerves failed to grow radially, the number of oligodendrocyte lineage cells was reduced compared with control nerves. However, the mitotic indices of progenitors and the percentage of oligodendrocytes undergoing programmed cell death were similar in control and transected optic nerves. Oligodendrocytes lacked their normal longitudinal orientation, developed fewer, shorter processes, and failed to form myelin in the transected nerves. These data indicate that normal densities of oligodendrocytes can develop in the absence of viable retinal ganglion axons, and support the possibility that axons assure their own myelination by regulating the number of myelin internodes formed by individual oligodendrocytes.     

The relationship between developing oligodendrocyte units and maturing axons during myelinogenesis in the anterior medullary velum of neonatal rats

Myelinogenesis was investigated in whole-mounted anterior medullary vela from rats aged postnatal day (P) 10–12, using double immunofluorescence labelling with Rip and anti-neurofilament 200 (NF200) antibodies, to identify oligodendrocytes and axons, respectively. A number of discrete phases of maturation of oligodendrocyte units were recognised. (1) Promyelinating oligodendrocytes co-expressed Rip and Myelin basic Protein and formed axonal associations, prior to ensheathment. (2) Transitional oligodendrocytes contained both ensheathing and non-ensheating processes. (3) Myelinating oligodendrocytes were established after a period of remodelling (in which non-ensheathing processes were lost), appearing as oligodendrocyte unit morphological phenotypes with a definitive number of incipient myelin sheaths. (4) Maturation of myelinating oligodendrocytes was defined as the establishment of internodal sheath lengths and the redistrubution of myelin basic protein from the cell somata and radial processes into the myelin sheaths only. Myelination was probably related to the maturational state of the axons, since it was initiated when the latter had attained a critical diameter of between ～0.2 and 0.4 μm, coincident with the expression of NF200. Oligodendrocyte differentiation and myelination of the AMV were asynchronous and multifocal, and at P10: (1) axons which were destined to be of the largest calibre in the adult AMV were already myelinated by early developing oligodendrocytes, whilst those which were destined to be the smallest calibre in the adult were unmyelinated, but ultimately became ensheathed by late developing oligoendrocytes; (2) axons were sequentially ensheathed by early developing myelinating oligodendrocytes and late developing promyelinating oligodendrocytes; (3) all axons were small calibre; (4) oligodendrocyte units exhibited polymorphism. Thus, the development of oligodendrocyte morphological phenotypes was not related solely to either the physical dimension of axon calibre at the time of ensheathment, nor oligodendrocyte birth dates.     

Oligodendrocyte precursor cell transplantation into organotypic cerebellar shiverer slices: a model to study myelination and myelin maintenance

Current in vitro models to investigate the consequence of oligodendrocyte-specific loss-of-function mutations on myelination are primarily limited to co-culture experiments, which do not accurately recapitulate the complex in vivo environment. Here, we describe the development of an in vitro model of myelination and myelin maintenance in which oligodendrocyte precursor cells are transplanted into organotypic cerebellar slice cultures derived from dysmyelinated shiverer mice. Compared to neuron-oligodendrocyte co-cultures, organotypic slices more closely mimic the environment in vivo, while utilizing a genetic background that allows for straight-forward identification of myelin generated by transplanted cells. We show at the ultrastructural level that the myelin generated by wild-type transplanted oligodendrocytes is compact and terminates in cytoplasmic loops that form paranodal junctions with the axon. This myelination results in the appropriate sequestering of axonal proteins into specialized domains surrounding the nodes of Ranvier. We also demonstrate the applicability of this approach for xenograft transplantation of oligodendrocyte precursor cells derived from rat or human sources. This method provides a time-efficient and cost-effective adjunct to conditional knockout mouse lines or in vivo transplantation models to study oligodendrocyte-specific loss-of-function mutations. Furthermore, the approach can be readily used to assess the effect of pharmacological manipulations on myelin, providing a tool to better understand myelination and develop effective therapeutic strategies to treat myelin-related diseases.     

Loss of ABCA8B decreases myelination by reducing oligodendrocyte precursor cells in mice

The myelin sheath, which is wrapped around axons, is a lipid-enriched structure produced by mature oligodendrocytes. Disruption of the myelin sheath is observed in several neurological diseases, such as multiple sclerosis. A crucial component of myelin is sphingomyelin, levels of which can be increased by ABCA8, a member of the ATP-binding cassette transporter family. ABCA8 is highly expressed in the cerebellum, specifically in oligodendroglia. However, whether ABCA8 plays a role in myelination and mechanisms that would underlie this role remain unknown. Here, we found that the absence of Abca8b, a mouse ortholog of ABCA8, led to decreased numbers of cerebellar oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes in mice. We show that in oligodendrocytes, ABCA8 interacts with chondroitin sulfate proteoglycan 4 (CSPG4), a molecule essential for OPC proliferation, migration, and myelination. In the absence of Abca8b, localization of CSPG4 to the plasma membrane was decreased, contributing to reduced cerebellar CSPG4 expression. Cerebellar CSPG4+ OPCs were also diminished, leading to decreased mature myelinating oligodendrocyte numbers and cerebellar myelination levels in Abca8b^?/? mice. In addition, electron microscopy analyses showed that the number of nonmyelinated cerebellar axons was increased, whereas cerebellar myelin thickness (g-ratio), myelin sheath periodicity, and axonal diameter were all decreased, indicative of disordered myelin ultrastructure. In line with disrupted cerebellar myelination, Abca8b^?/? mice showed lower cerebellar conduction velocity and disturbed locomotion. In summary, ABCA8 modulates cerebellar myelination, in part through functional regulation of the ABCA8-interacting protein CSPG4. Our findings suggest that ABCA8 disruption may contribute to the pathophysiology of myelin disorders.     

Adhesion molecules in the regulation of CNS myelination

Myelination is necessary both for rapid salutatory conduction and the long-term survival of the axon. In the CNS the myelin sheath is formed by the oligodendrocytes. Each oligodendrocyte myelinates several axons and, as the number of wraps around each axon is determined precisely by the axon diameter, this requires a close, highly regulated interaction between the axons and each of the oligodendrocyte processes. Adhesion molecules are likely to play an important role in the bi-directional signalling between axon and oligodendrocyte that underlies this interaction. Here we review the current knowledge of the function of adhesion molecules in the different phases of oligodendrocyte differentiation and myelination, and discuss how the properties of these proteins defined by other cell biological systems indicates potential roles in oligodendrocytes. We show how the function of a number of different adhesion and cell-cell interaction molecules such as polysialic acid neural cell adhesion molecule, Lingo-1, Notch, neuregulin, integrins and extracellullar matrix proteins provide negative and positive signals that coordinate the formation of the myelin membrane. Compiling this information from a number of different cell biological and genetic experiments helps us to understand the pathology of multiple sclerosis and direct new areas of research that might eventually lead to potential drug targets to increase remyelination.     

Axo-Glia Interaction Preceding CNS Myelination Is Regulated by Bidirectional Eph-Ephrin Signaling

In the central nervous system, myelination of axons is required to ensure fast saltatory conduction and for survival of neurons. However, not all axons are myelinated, and the molecular mechanisms involved in guiding the oligodendrocyte processes toward the axons to be myelinated are not well understood. Only a few negative or positive guidance clues that are involved in regulating axo-glia interaction prior to myelination have been identified. One example is laminin, known to be required for early axo-glia interaction, which functions through α6β1 integrin. Here, we identify the Eph-ephrin family of guidance receptors as novel regulators of the initial axo-glia interaction, preceding myelination. We demonstrate that so-called forward and reverse signaling, mediated by members of both Eph and ephrin subfamilies, has distinct and opposing effects on processes extension and myelin sheet formation. EphA forward signaling inhibits oligodendrocyte process extension and myelin sheet formation, and blocking of bidirectional signaling through this receptor enhances myelination. Similarly, EphB forward signaling also reduces myelin membrane formation, but in contrast to EphA forward signaling, this occurs in an integrin-dependent manner, which can be reversed by overexpression of a constitutive active β1-integrin. Furthermore, ephrin-B reverse signaling induced by EphA4 or EphB1 enhances myelin sheet formation. Combined, this suggests that the Eph-ephrin receptors are important mediators of bidirectional signaling between axons and oligodendrocytes. It further implies that balancing Eph-ephrin forward and reverse signaling is important in the selection process of axons to be myelinated.     

Myelin sheath survival after guanethidine-induced axonal degeneration

Membrane-membrane interactions between axons and Schwann cells are required for initial myelin formation in the peripheral nervous system. However, recent studies of double myelination in sympathetic nerve have indicated that myelin sheaths continue to exist after complete loss of axonal contact (Kidd, G. J., and J. W. Heath. 1988. J. Neurocytol. 17:245-261). This suggests that myelin maintenance may be regulated either by diffusible axonal factors or by nonaxonal mechanisms. To test these hypotheses, axons involved in double myelination in the rat superior cervical ganglion were destroyed by chronic guanethidine treatment. Guanethidine-induced sympathectomy resulted in a Wallerian-like pattern of myelin degeneration within 10 d. In doubly myelinated configurations the axon, inner myelin sheath (which lies in contact with the axon), and approximately 75% of outer myelin sheaths broke down by this time. Degenerating outer sheaths were not found at later periods. It is probably that outer sheaths that degenerated were only partially displaced from the axon at the commencement of guanethidine treatment. In contrast, analysis of serial sections showed that completely displaced outer internodes remained ultrastructurally intact. These internodes survived degeneration of the axon and inner sheath, and during the later time points (2-6 wk) they enclosed only connective tissue elements and reorganized Schwann cells/processes. Axonal regeneration was not observed within surviving outer internodes. We therefore conclude that myelin maintenance in the superior cervical ganglion is not dependent on direct axonal contact or diffusible axonal factors. In addition, physical association of Schwann cells with the degenerating axon may be an important factor in precipitating myelin breakdown during Wallerian degeneration.     

Initiation of sodium channel clustering at the node of Ranvier in the mouse optic nerve

Ion fluxes in mammalian myelinated axons are restricted to the nodes of Ranvier, where, in particular, voltage-gated Na⁺ channels are clustered at a high density. The node of Ranvier is separated from the internode by two distinct domains of the axolemma, the paranode and the juxtaparanode. Each axonal domain is characterized by the presence of a specific protein complex. Although oligodendrocytes and/or myelin membranes are believed to play some instructive roles in the organization of axonal domains, the mechanisms leading to their localized distribution are not well understood. In this paper we focused on the involvement of myelin sheaths in this domain organization and examined the distribution of axonal components in the optic nerves of wild type, hypomyelinating jimpy mice and demyelinating PLP transgenic mice. The results showed that the clustering of Na⁺ channels does not require junction-like structures to be formed between the glial processes and axons, but requires mature oligodendrocytes to be present in close vicinity.     

Differentiation and Death of Premyelinating Oligodendrocytes in Developing Rodent Brain

Previous studies have indicated that newly formed oligodendrocytes are dynamic cells whose production, survival, and differentiation depend upon axonal influences. This study has characterized the appearance and fate of newly formed oligodendrocytes in developing rat brain. Oligodendrocytes appear in predictable locations and radially extend DM-20–positive processes that cover 80-μm domains in the cortex and 40-μm domains in the corpus callosum. These premyelinating oligodendrocytes have one of two fates: they myelinate axons or degenerate. Between 7 and 21 d after birth, ~20% of premyelinating oligodendrocytes identified in the cerebral cortex were degenerating. Oligodendrocytes that ensheathed axons expressed and selectively targeted proteolipid protein to compact myelin and did not degenerate. These observations support the hypothesis that axonal influences affect oligodendrocyte survival, differentiation, and expression of proteolipid protein gene products.     

Correlation between electrophysiological properties,morphological maturation,and olig gene changes during postnatal motor tract development

This study investigated electrophysiological and histological changes as well as alterations of myelin relevant proteins of descending motor tracts in rat pups. Motor‐evoked potentials (MEPs) represent descending conducting responses following stimulation of the motor cortex to responses being elicited from the lower extremities. MEP responses were recorded biweekly from postnatal (PN) week 1 to week 9 (adult). MEP latencies in PN week 1 rats averaged 23.7 ms and became shorter during early maturation, stabilizing at 6.6 ms at PN week 4. During maturation, the conduction velocity (CV) increased from 2.8 ± 0.2 at PN week 1 to 35.2 ± 3.1 mm/ms at PN week 8. Histology of the spinal cord and sciatic nerves revealed progressive axonal myelination. Expression of the oligodendrocyte precursor markers PDGFRα and NG2 were downregulated in spinal cords, and myelin‐relevant proteins such as GalC, CNP, and MBP increased during maturation. Oligodendrocyte‐lineage markers Olig2 and MOG, expressed in myelinated oligodendrocytes, peaked at PN week 3 and were downregulated thereafter. A similar expression pattern was observed in neurofilament M/H subunits that were extensively phosphorylated in adult spinal cords but not in neonatal spinal cords, suggesting an increase in axon diameter and myelin formation. Ultrastructural morphology in the ventrolateral funiculus (VLF) showed axon myelination of the VLF axons (99.3%) at PN week 2, while 44.6% were sheathed at PN week 1. Increased axon diameter and myelin thickness in the VLF and sciatic nerves were highly correlated to the CV (rs > 0.95). This suggests that MEPs could be a predicator of morphological maturity of myelinated axons in descending motor tracts. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 73: 713–722, 2013     

Oligodendrocyte Birth and Death following Traumatic Brain Injury in Adult Mice

Oligodendrocytes are responsible for producing and maintaining myelin throughout the CNS. One of the pathological features observed following traumatic brain injury (TBI) is the progressive demyelination and degeneration of axons within white matter tracts. While the effect of TBI on axonal health has been well documented, there is limited information regarding the response of oligodendrocytes within these areas. The aim of this study was to characterize the response of both mature oligodendrocytes and immature proliferative oligodendrocyte lineage cells across a 3 month timecourse following TBI. A computer-controlled cortical impact model was used to produce a focal lesion in the left motor cortex of adult mice. Immunohistochemical analyses were performed at 48 hours, 7 days, 2 weeks, 5 weeks and 3 months following injury to assess the prevalence of mature CC-1⁺ oligodendrocyte cell death, immature Olig2⁺ cell proliferation and longer term survival in the corpus callosum and external capsule. Decreased CC-1 immunoreactivity was observed in white matter adjacent to the site of injury from 2 days to 2 weeks post TBI, with ongoing mature oligodendrocyte apoptosis after this time. Conversely, proliferation of Olig2⁺ cells was observed as early as 48 hours post TBI and significant numbers of these cells and their progeny survived and remained in the external capsule within the injured hemisphere until at least 3 months post injury. These findings demonstrate that immature oligodendrocyte lineage cells respond to TBI by replacing oligodendrocytes lost due to damage and that this process occurs for months after injury.     

Death receptor 6 negatively regulates oligodendrocyte survival, maturation and myelination

Survival and differentiation of oligodendrocytes are important for the myelination of central nervous system (CNS) axons during development and crucial for myelin repair in CNS demyelinating diseases such as multiple sclerosis. Here we show that death receptor 6 (DR6) is a negative regulator of oligodendrocyte maturation. DR6 is expressed strongly in immature oligodendrocytes and weakly in mature myelin basic protein (MBP)-positive oligodendrocytes. Overexpression of DR6 in oligodendrocytes leads to caspase 3 (casp3) activation and cell death. Attenuation of DR6 function leads to enhanced oligodendrocyte maturation, myelination and downregulation of casp3. Treatment with a DR6 antagonist antibody promotes remyelination in both lysolecithin-induced demyelination and experimental autoimmune encephalomyelitis (EAE) models. Consistent with the DR6 antagoinst antibody studies, DR6-null mice show enhanced remyelination in both demyelination models. These studies reveal a pivotal role for DR6 signaling in immature oligodendrocyte maturation and myelination that may provide new therapeutic avenues for the treatment of demyelination disorders such as multiple sclerosis.     

Morphological differentiation of oligodendrocytes requires activation of Fyn tyrosine kinase.

In the central nervous system, myelination of axons occurs when oligodendrocyte progenitors undergo terminal differentiation and initiate process formation and axonal ensheathment. Although it is hypothesized that neuron-oligodendrocyte contact initiates this process, the molecular signals are not known. Here we find that Fyn tyrosine kinase activity is upregulated very early during oligodendrocyte progenitor cell differentiation. Concomitant with this increase is the appearance of several tyrosine phosphorylated proteins present only in differentiated cells. The increased tyrosine kinase activity is specific to Fyn, as other Src family members are not active in oligodendrocytes. To investigate the function of Fyn activation on differentiation, we used Src family tyrosine kinase inhibitors, PP1 and PP2, in cultures of differentiating oligodendrocyte progenitors. Treatment of progenitors with these compounds prevented activation of Fyn and reduced process extension and myelin membrane formation. This inhibition was reversible and not observed with related inactive analogues. A similar effect was observed when a dominant negative Fyn was introduced in progenitor cells. These findings strongly suggest that activation of Fyn is an essential signaling component for the morphological differentiation of oligodendrocytes.     

Neurotransmitter-Triggered Transfer of Exosomes Mediates Oligodendrocyte–Neuron Communication

Reciprocal interactions between neurons and oligodendrocytes are not only crucial for myelination, but also for long-term survival of axons. Degeneration of axons occurs in several human myelin diseases, however the molecular mechanisms of axon-glia communication maintaining axon integrity are poorly understood. Here, we describe the signal-mediated transfer of exosomes from oligodendrocytes to neurons. These endosome-derived vesicles are secreted by oligodendrocytes and carry specific protein and RNA cargo. We show that activity-dependent release of the neurotransmitter glutamate triggers oligodendroglial exosome secretion mediated by Ca²⁺ entry through oligodendroglial NMDA and AMPA receptors. In turn, neurons internalize the released exosomes by endocytosis. Injection of oligodendroglia-derived exosomes into the mouse brain results in functional retrieval of exosome cargo in neurons. Supply of cultured neurons with oligodendroglial exosomes improves neuronal viability under conditions of cell stress. These findings indicate that oligodendroglial exosomes participate in a novel mode of bidirectional neuron-glia communication contributing to neuronal integrity.